Iycee Charles de Gaulle Summary The Concept Of Contamination During Cryopreservation Biology Essay

The Concept Of Contamination During Cryopreservation Biology Essay

It has been examined that the liquid N is chief beginning to pollute the 70 % sucrose solution. The 70 % sucrose solution check home bases are possible for taint during the cryopreservation stairss. The method of experiment was depends on the contaminated liquid N. The crystals of sucrose hemi-heptahydrate are used to pollute the liquid N. These sucrose hemi-heptahydrates are stable in liquid N.

In this the experiment method used is, the 70 % sucrose solution assay home bases exposed to contaminated liquid N bluess for approximately 4 hours, and these sucrose check home bases are maintained at different distances are 100cm, 200cm, and 300cm. The open sucrose check home bases are cryopreserved at -20oC for 5 to 15 yearss. It was demoing that merely liquid N bluess are used as control. The 70 % sucrose solution home bases are placed at different distances to liquid N.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

Expose the control samples for 4 hours and cyropreserve it for 5 to 15 yearss. After cryopreservation of sucrose check home bases observed and the consequences are noted as, in control samples there are no crystal formation seen. While those treated sucrose check home bases demoing farinaceous crystals. The treated check home bases which are closing ( 100cm ) to the liquid N are holding more crystals so the saccharose home bases which are placed at a distance of 200cm and 300cm. Here least crystals are observed in check home bases which are at 300cm from the liquid N flask.

The distance between the liquid N and sucrose solution home bases, the exposure clip period of sucrose home bases to the liquid N, and the concentration of sucrose hemi heptahydrate used are conforming the purpose of this experiment. Harmonizing to these consequences the liquid N is playing major function in taint during cryopreservation.Cryopreservation is a procedure in which cells or the whole tissues incorporating a figure of cells are preserved by chilling it to a low temperature, about to a low temperature of 77kA°C or -196A°C.

In this several techniques are used in which micro-organism, tissues and embryos are separated from each other. The chief beginning of life in a life cell is H2O in chemical signifier. So to hold the metamorphosis in cell, the H2O is converted into the ice at different rates during the chilling procedure. During this procedure of slow chilling, the freeze occurs on the external parts of the cells before the intracellular parts.

When the ice is formed on the external sides, the H2O is removed from the extracellular environment, which leads to a osmotic instability across the cell membrane by which the H2O from inside the cell is besides migrated out.So the addition in this solute concentration on both inside and outside of a cell leads to the endurance of the cell. There are many compounds have tested as cryo-protective agents but glycerin and DMSO is best consequence in the cryopreservation. DMSO is widely used for the desiccation of cells to intracellular freeze. The cryo-protective agents are depends upon the type of the cell to be conserved glycerol normally less toxic so DMSO. In some instance, during/ after the storage or cryo-preservation of cells, the taint takes topographic point which can of any type depending upon the nature and characteristics of the contaminating agent.

1.6. Contamination:

Contamination in a biological scientific discipline by and large means an inadvertent or knowing debut of foreign stuffs ( polluting agents ) which can earnestly falsify the consequences of experiments with the stored samples. There are different types of polluting agents which are H2O, visible radiations, brooders, viruses, bacterium ‘s, invertebrates and mycoplasma.

There are different types of taints they are:

1.6.1. Environmental Contamination:

It has been noticed that 1-10 % samples of cells and tissues gets contaminated during the clinical or laboratory use and in some instance besides when infective beings are potentially stray ( Lieby, et al.,1997 ) . Most the taint instances in the research labs have through bacteriums ‘s and fungus from different beginnings, for e.g. composition board boxes used to hive away lab consumables.

Sinks and penstocks in the lab, which can do fungous growing may besides be a ground of taint in labs, so the H2O baths and the cold suites must be cleaned and sanitised on a regular basis. The other services such as supplied distilled H2O used to execute assorted operation in the labs, can besides be a instance of taint because, the contaminated distilled H2O sometimes contains bacteriums that releases Endotoxins or other beings such as Acholeplasma which can easy steal through the bacterium filters. Most of all the deficiency of preparation and aid of working in the lab can besides take to the taint.


6.1.1. Storage quality:

The topographic point or the medium used to hive away the samples can besides take to the taint.

For illustrations if a sample is stored in a medium, which antecedently contained a solution of heavy metals or organic compounds can besides be a beginning of taint ( Fountain, et al. , 1997 ) . The quality of the storage medium can besides be a ground of taint because if the quality of medium is non good so it can take to the escape of the sample or the viruses which may pollute the other samples.


2. Contamination through brooders:

The brooders are frequently considered to be the major beginning of taint in both biological and chemical manner. The mixture of gases which are suffused through brooders can incorporate toxic drosss such as oils or other gases, which may be used in the same storage cylinder or armored combat vehicles. Incubator job is really rare in medical class gases but more common in less expensive industrial class gases.

Adventitious Mediator Cell Cultures:

The intent of in vitro cell civilization for tolerant substance and the usage of cell lines and their merchandises are increasing quality of curative engineering. Cell lines of mycoplasma taint will distribute quickly to other civilizations if non quickly isolated and ever contributes to constant infection that may alter the quality of the cells. ( Aula.p et al.1967 ) . Cheap and rapid techniques are available in the cell lines for mycoplasma. ( Mowles et al.1990 ) .

Cell lines and their merchandises carry contaminations which represent a serious menace to human wellness such as lymphocytic choriomeningitis ( LCMV ) and Hantavirus. ( Nicklas et al.1993 ) . In general cell lines have to prove for possible pathogens based on those likely to originate from the host species and beginning of tissue that trial cells derived from human beginnings for human pathogens ( Frommer et al.

, 1992 ) .


Microbial taint through cryogenic equipments:

It by and large means an inclusion or growing of harmful micro-organisms such as clostridia botulinus. This growing can be on anything such as nutrient or stored sample doing the insecure or fresh for future intent. Three major types of cryogenic equipment have been contaminated with nuisance micro-flora are: Controlled rate deep-freeze, dry shippers, and storage cryo-tanksA cryogenic application includes liquified gases, such as liquid N and liquid He. Liquid N is most normally used in cryogenic procedure and it is besides lawfully corruptible throughout the universe. Liquid He is besides used in some instance because of its nature of leting lowest come-at-able temperature comparison to others.

Grout and Morris the two good known scientists in 2008 conducted a trial in which they dosed a majority sum of liquid N with Sclerotium which is a fungous pathogen in liquid signifier, to chill a programmable liquid N deep-freeze. After one chilling tally, when the cryogenic instrument was tested, the whole surface showed positive which signified the presence of taint in the instrument. The experiment conducted by Grout and Morris proved that the pathogens can last but by polluting the liquid N and the cryogenic device.


1. Dry Shippers:

In 2005 another scientist Bielanski by experimentation contaminated the samples of seeds and embryos and besides the two different theoretical accounts of dry shippers. 270,000embryos and 264million doses of bovid seeds were cryo-preserved for the used on the twelvemonth 2002. ( Polge et.al 1947 ) homo gametes are non available in the cryopreservation so infected with hepatitis viruses and HIV. Germplasm stored in the liquid N stage of cross taint suited to container breakage at 196 A°C in liquid N. Then, he tested the tracts of bacterial taint during the transmittal from and between. It found that the dry shippers were contaminated to germplasm, some of the cryo-preserved germplasm were contaminated while some were non-contaminated and a stock civilization of infective agents & A ; germplasm.

After that he stored the contaminated and not contaminated samples of embryos and seeds ‘s for about 7 yearss in a unfastened container with liquid N in vapour stage. After 7 yearss he made a trial of bacterial taint but no grounds of cross-contamination from either contaminated to non-contaminated samples or between contaminated Dewar ‘s to germplasm. This led to a decision that the vapour stage of liquid N in the dry shippers can be used for the long term storage of germplasm, even though the shippers or some of the samples are contaminated.Cryogenic equipment maker have developed new merchandises to run into the demands of users and practicians who requires double-skin the samples that can go either accidently or purposefully in direct contact with liquid stage liquid N. Transmission of three bacterial and two viral micro-organisms was cryopreserved from embryos and seeds by vapour stage of liquid N stored in the dry shipper. The absorbent forces back to the humidness absorb liquid N of vapour stage at-150 A°C in the storage chamber without the liquid N release. Bacterial and viral agents was detected in all station melt samples of embryos and seeds exhibited to the agents to cryopreservation and the civilization stocks used as a contaminations under the storage of vapour stage.

There is no transmittal of agents between contaminated and not contaminated containers stored in the vapour stage of liquid N. The concentrations of bacterial and viral civilizations of the vapor shippers was exhibited possible through storage and preservation of germplasm. The micro-organisms used in the two gm negative and one gm positive bacterial agent on the signifier individual strand RNA and dual criterion DNA virus. ( w.c.d.

Hare et.al 1985 ) .The cryo-protective agents of germplasm infected with hepatitis C and human herpes viruses. E.g. catching spongiform brain disorder ‘s ( TSE ) can be harmful by LN bluess.

1.6.3. Different microbic contamination commanding in cryo-banks/tanks:

This process includes objecting of hazards related to the cross-contamination between samples in cryo-tanks. It mostly involves the proficient and direction safeguards which are double-contaminant, avoiding of direct contacts with liquid N, and the storage of vapour signifier of liquid N.

There are several classs of microbic taints. Virus and virusoids:

To cognize the issue of microbic taint, the works viruses are stored and used which thought to be utile. In the works viruses besides the Deoxyribonucleic acid viruses are more stable so the RNA viruses.

Many of the works viruses can be stored in dehydrated host tissues at around 4A°C for several old ages ( Gould, 1995 ) . During the taint procedure it is advised that, the viruses when cryo-preserved should be encased in dual certain tubings. These observations on the works viruses have put frontward an of import consideration in cryo-banking of works cells which carries virus infections during the clip of cryopreservation ( Hatta and Francki, 1981 ) .

To turn out this construct many people came up with their theories and experiments.


3.2. Confirmation based hazard appraisal of virus transmittal via cryobanks:

Before many scrutinies were held on grounds of know about the virus transmittal utilizing cryobanks. In 1995 Tedder came up with the first grounds for which she performed an experiment. The experimental information was consist of bone marrow and blood merchandises from wickedness multi transfused patients which in stored in cryostorage for about 25months these samples were stored in cryobanks utilizing the cryobags. The escape of the cryobag which is used to hive away the bone marrow of the first patient gets foremost infected with the hepatitis B virus and so it contaminates full armored combat vehicle and its content so the concluding result was the patient acquiring infected. Due to this Tedder provided same suggestions about the stairss to be followed before cryopreserving any samples.1 Tissue of the givers must be screened to observe any blood borne viral infections.

2 Samples should be stored in dual containers.In 1997 fountain stated that both vapors and liquid stage N can besides do microbiological taint Clarke in 1999 besides came up with same similar instances of cross taint which was caused by the storage of human tissues. But in 2000 Bielanski came up with something wholly different. This method of cryopreservation included extremist rapid or vitrification of protocols which required direct contact of stop deading medium incorporating the tissues with liquid N. The experiment included used of bovine embryos trial and a scope of different animate beings human pathogen viruses to by experimentation infect embryos.

The whole thing is cryopreserved in a vitrification solution which contains DMSO, ethene glycerin and saccharose. Both septic and clean samples were cryopreserved in different cryocontainers with different sealant capacities. These cryocontainers are stored in the same Dewar in the liquid stage of liquid N for around 3-5 hebdomads.

The chief determination of Bielanski concluded a menace of embryos going contaminated from stop deading during the cryopreserved procedure in unfastened containers so exposing to liquid N in liquid stage advised certain stairss to be followed.1. Make the usage of container with which is hermetically sealed.2. Avoiding the direct contact between liquid N and freeze medium3. Always hive awaying contaminated and not contaminated samples in different armored combat vehicles.

In 2003 Bielanski expanded his survey which signified no grounds of the transportation of bovine virus between contaminated and not contaminated embryos and seed ‘s. When stored in certain plastic straws with liquid N for a clip period of 6-35years. ( Berz in 2007 ) advised some degrees of containments which are necessary for avoiding viral infection in cryopreservation of hematopoietic root cells. To show this cross taint he besides recommend dual bagging the samples in protective arms and infected samples which should be placed in separate cryotanks. In another incident ( Husebekk et al. , 2004 ) reported peripherals of blood primogenitor cells ( PBPCs ) which were taken from a patient but during the procedure of stop deading the cell suspension by chance leaked on the surface of cryobags. Then these bags were transferred to a 2nd protective envelop, dispensed into a steel cassette and placedin a armored combat vehicle which contains liquid N in vapour stage.

This armored combat vehicle already contains other samples tested virus-free.

Abioticand biotic hazard appraisals for virus transmittal in cryobanks:

Based on the carnal medical and biotechnical literature the transmittals were needed to be clarified as the degree of hazard associated with cryogenic transmittal of viral micro-organisms. The hazard Level accepted by different checkups, animate beings, and biotechnology communities. Although different people advised these degrees of safeguard that should be kept in head before cryopreserving any samples, the possible hazards for cross taint of phytological viruses and virusoids in both new and already established cryorepositories will be determined by biotic and abiotic factors. Each and every viruses or virusoids have their ain degree of hazard viruses are wholly dependent on the substance to substance transmittal taint and have complex transmittal infection procedure which may be considered as a lower hazard class with their potency of cross infection via cryogenic paths ( Vander Want and Dijksta, 2006 ) . The hazard of virus transmittal occurs through assorted cryogenic paths. These paths can be expected to be higher for those works pathogens which are transmitted from direct contact and natural wounding, in the instance for murphy virus.


4. Mycoplasma:

Mycoplasm ‘s are the smallest free life retroflexing beings which are found in mature. These beings have a certain simple construction incorporating proteins DNA RNA and enzymes.

These are besides classified as Mollicutes because they lack cells walls and are bounded by a ternary bed unit membrane. Whereas phytoplasma is a term which is applied to mycoplasma type beings ( Namba, 2002 ; Lee et al. , 2000 ) . These beings are ab initio designated by the systematic group of campaigners ‘ phytoplasm species which are found in workss such beings can take to serious agribusiness diseases. Phytoplasmas are transmitted into the workss through vectors and can be spreaded by infecting the workss by grafting its healthy works.

Mycoplasma infections in animate being and human cells cryobanks can take to a black effect.

1.7. Prevention taken to avoid cryo-preservation hazards:

1.7.1. Controlled-rate and slow freeze:

This technique was established in early 1970s. In 1984, it enabled the first human embryo frozen birth.

Then all over the universe these machines for stop deading biological samples sample came into usage for different programmable stairss and controlled rates for homo, animate being and cell biological science. In this a sample, is frozen down to better continue it for eventual nature, before it is profoundly frozen or cryo-preserved in liquid N. These machines are used for hive awaying and stop deading the samples like tegument, blood merchandises, sperm, embryos, root cells and tissue saving at many topographic points such as research labs and infirmaries. It has been proved in several surveies that the frozen embryos stored utilizing slow-freezing techniques are someway better than other technique used in cryopreservation. These surveies besides revealed that the usage of frozen embryos instead than fresh embryos reduces the hazard of abortion and premature bringing of the trials and experiments.


2. Vitrification:

It is a technique which includes the benefits of cryopreservation without damaging the samples because of the ice crystal formation. Every cell contains H2O into it for life.

When the tissue is cooled below its freeze point, the H2O molecules get together and organize a grouping crystal. When the H2O gets wholly frozen, the ice squeezes other molecules inside the tissue in a detrimentally concentrated solution. First the ice is formed on the outer portion of the cells and continues turning of ice over the cell causes it dehydrate and psychiatrist. Then the cells are left damaged and squashed between ice crystals. Then the chemicals which are known as cryoprotectants are added to the H2O to forestall H2O molecules from garnering together to organize ice.

Alternatively of freeze, the molecules merely travel slower and slower as they are cooled and eventually at a temperature below -100C molecules are turned into solid without stop deading so this is said to be “ glassy ” . Again the cryo-protectants are added to the cells before the sample is profoundly cooled. In this procedure there is no harm held to the cells because during the chilling no ice is formed inside the cells and eventually the biological clip of the cell is stopped. These samples can be stored for comparatively long periods in this solid signifier without the concern of air taint or H2O taint.


Contamination of testing root cells:

The stuffs are tested for contaminations free. By testing bacterial choice media, the most of taint which are due to bacteriums can be easy discovered.To inoculate the flask that consists of 10 ml civilization medium, one phial is used. The presence of turbidness in which white coloring material indicates the bacterial taint after two hebdomads incubated at 37A±c.The ice deposit which is accumulate in the armored combat vehicles of liquid N during storage can do the taint of samples ( Piasecka and serafin,1972 ) .Even though in the cryopreservation of sperms in the fish is non affect the sterile techniques, the usage of unfertile techniques plays a major function for commanding the taint ( Stoss,1983: Lahnsteiner et al,2000: Chao and Liao 2001 ) . Beilanski ( 1997 ) explained that sample of embryos that are stored in the contamination liquid N are tested negative for observing the presence of BVDV and BAV1 viruses.

Hence for forestalling taint it is recommended that thawed straws are cut by utilizing unfertile scissors and scrapped blade. Same like the cross taint in the long term storage of sample can be prevented by utilizing high security straws and every bit good as separate liquid N. Piasecka-Serafin M, 1972, the consequence of deposit accumulated in containers under experimental conditions on the infection of seeds stored straight stored in liquid N, Bull Acad Pol Sci Biol, 20, 263-7.

Chapter 2


2.1 Sucrose:

Sucrose was a compound which contains more or big figure of Cs in its molecular expression. The molecular expression of saccharose is C12H22O11. Sucrose was high category of chemical compound member ( Spencer et.al, 2004 ) .

The international brotherhood of pure and applied chemical science terminology ( IUPAC ) of saccharose is I?-D-fructofuranosyl- ( 2a†’1 ) -I±-D-glucopyranoside. The common name of saccharose was table sugar, by and large sugar. It is besides called sucrose. The physical nature of the saccharose was crystalline sugar, white in coloring material, odorless. And the saccharose was sweet in gustatory sensation. It by and large used for human nutrition. Sucrose was disaccharide and it was obtained by the combination of two monosaccharoses which are D-Glucose and D-Fructose. The bond formed between these two monosaccharoses was called glycosidic linkage.

The molecular expression of glucose and fruit sugar is C6H12O6.File: Sucrose CASCC.pngFigure: 2.1. Molecular Structure of SucroseThe hydrolysis of sucrose occurs really easy, in presence of accelerator or acids the sucrose interruption down into glucose and fructose. In this procedure the break-down of glycosidic linkage occurs. By the hydrolysis of disaccharide become two monosaccharoses ( Beevers and MeDonlads, 1952 ) .

2.1.1. Preparation of 70 % sucrose solution:

The 70grams of saccharose was weighed in weighing machine by utilizing spatula.

These sucrose crystals were taken into a conelike flask or a bottle that incorporating with 100 milliliter of distilled H2O. Shake this conelike flask and splash with scaremonger. Heat the solution for approximately 4 to 5 proceedingss in microwave to fade out the sucrose crystals in distilled H2O. Shop this 70 % of sucrose solution at 4A°C for farther usage.The 70 % sucrose solution can besides fix as follows: 70ml of sucrose indicates 70 % . So 70 gms of sucrose crystals dissolved in 100 milliliter of distilled H2O.

The 49 gms of sucrose crystals are taken to fade out in 70ml of distilled H2O for the readying of 70 % sucrose solution.i.e. 70 gms sucrose — — — — — — – & gt ; 100ml’x’grams sucrose — — — — — — – & gt ; 70mlTherefore: this gives 49 gms.

F: PhotosxDSC_0282.jpgFigure: 2.2. 70 % of sucrose solution check home base


2. Sucrose Hemi Heptahydrate:

An anhydrous signifier of sucrose crystallizes into two signifiers that are sucrose hemi pentahydrate and sucrose hemi heptahydrate. These crystallizes signifiers of sucrose occurs at really low temperature that was -34A°C ( Young and Jones ) . The molecular expression of sucrose hemi heptahydrate is C12H22O11.3.

5.H.Sub.2O. This heptahydrate is phase II saccharose hydrate. This hemi heptahydrate signifiers at the temperature of -9.

5oC. The liquescent temperature of sucrose hemi heptahydrate is +27.8oC. It is used to pollute the liquid N ( Young and Francis ) .

2.3. Liquid Nitrogen:

The liquid province of N gas occurs at really low temperature was called liquid N. It was colorless liquid. LN2 and LN are the abbreviation for the liquid N. The boiling point or the liquid N furuncles at the temperature of -196oC. The liquid N was cryogenic liquid, so liquid N was called as cryogenic agent that means it surveies at really low temperature.

Liquid N freezes quickly, it used to stop dead populating tissues in fraction of clip. Liquid N can easy change over into solid by puting in vacuum chamber. The freezing temperature of liquid N was -210oC. Liquid N was a beginning used to transport the life tissues and variety meats. Liquid N was besides used to continue for long period of clip without any hurt. Liquid N was a beginning of dry N gas.

LN was used to cyropreserve of life tissues, carnal cells, works cells, and blood, sperms, egg and biological samples for long clip. The enlargement ratio of N from liquid to gas was 1:694. By and large the N was non-toxic, odourless. It was an inert gas and the N was non flammable, the liquid N was hydrophobic ( Umrath 1974 ) .

2.4. Dewar Flask:

Dewar flasks were used to hive away liquids and it was used to hive away the liquid N in little and big measures. Generally liquid N shops in vacuity infinite that nowadays between the inner and outer shells of the Dewar flasks.

Durable and aluminum ( light weight ) are used to do the outer shell of the Dewar flask. Insulation is used to make full the vacuity infinite that nowadays between the outer and inner shell of the Dewar flask. The necktube nucleus is besides used to cut down or command the loss or tax write-off of liquid N. The borosilicate glass was used to keep the liquid N service. The vaporization of liquid N was controlled or minimizes by utilizing the polythene in Dewar flask.

Degree centigrades: UserskrishDesktopcarrying_flask_typeb-e.gifFigure: 2.3. Dewar flask

2.5. Method of Experiment:

The undermentioned method is used to rating of taint after exposing to contaminated liquid N with sucrose hemi heptahydrate for about 4 hours clip period.

The method was helpful to turn out hypothesis and this method of experiment screens aim and objective. By this hypothesis it helps scientifically for farther usage in cryopreservation.

2.5.1. Preparation of Negative control:

In 100ml of distilled H2O add 70gm of sucrose crystals or in 70 milliliter of distilled H2O add 49gm of sucrose crystals for readying of 70 % sucrose solution.

Stir the solution to fade out saccharose or heat for approximately 4 to 5 proceedingss in microwave. Pour the 70 % sucrose solution into 9 clean and fresh labelled Petri dishes. In another side transportation 1liter to 2liter of liquid N into little Dewar flask and maintain 3 Petri home bases near the liquid N with distance of 100cm. Use another 3 Petri dishes, maintain these with distance of 200cm.

And utilize staying 3 Petri dishes maintain them with the distance of 300cm. Expose these all 9 Petri dishes to the liquid N bluess for approximately 4 hours. After 4 hours near these Petri home bases with Parafilm and screen with cleaving movie subsequently preserved these labelled Petri dishes at -20oC for about 15 yearss.

These 9 labelled Petri dishes are uses as negative control for the experiment. Preparation of Positive control:

The positive control was used to analyze the hypothesis of the experiment. The negative control was used to compare the consequences of the positive control. In this sucrose hemi heptahydrate was used to pollute the liquid N.

The contaminated liquid N was exposed to the 70 % sucrose solution assay home bases for approximately 4 hours at different distances of 100cm, 200cm and 300cm. After exposure of contaminated liquid N to the sucrose home bases, cryopreserved them for 5 to 15 yearss at a temperature of -20oC. These assay home bases are used as positive control for the experiment. Evaluation of taint at different distances:

Pollute the liquid N with the sucrose hemi heptahydrate.

In this experiment method 0.5gm to 3gm concentration of sucrose hemi heptahydrate crystals is used to pollute the liquid N. The contaminated liquid N was used to analyze this experiment. Add 70 % sucrose solution ( that stored in 4oC ) to Petri dishes for the experiment. Label all the Petri dishes. Place three of these labelled Petri dishes towards the contaminated liquid N with the distance of 100cm. Use another three Petri dishes at a distance of 200cm and likewise usage another three Petri dishes at a distance of 300cm.

Expose these 9 Petri dishes to liquid N bluess which is contaminated by sucrose hemi heptahydrate about 4 hours. After 4 hours exposing these 70 % sucrose solution Petri dishes are closed with it lid and seal it with Parafilm. Then wrap the certain Petri dishes with Clingfilm. And continue these all Petri dishes at the temperature of -20oC for approximately 15days. After 5days observe these cryopreserved Petri dishes every twenty-four hours and do a note. Repeat the same experiment for approximately 5 to 7 times, and observe the consequence at all the times and do a note it. F: PhotosxDSC_0107.

jpgFigure: 2.4. Sucrose assay home bases at different distances to liquid N


3. Data Collection:

After 5days observe these cryopreserved control Petri dishes every twenty-four hours, and collect the information by detecting Petri dishes. Similarly cod informations from the treated Petri dishes every bit good. Roll up the informations from all the Petri dishes that different distance of 100cm, 200cm and 300cm. For every perennial experiment control besides needed to analyze.

Chapter 3


To measure the taint during cryopreservation, the 70 % sucrose solution was prepared and treated with contaminated liquid N bluess that contaminated with sucrose hemi heptahydrate.

Consequences observed in negative control:

Control was used to measure or compare the consequences with positive control. 70 % sucrose solution was exposed to liquid N that without contaminated sucrose hemi heptahydrate, these Petri dishes are used as the negative control for the treated 70 % sucrose solution samples. These control and treated sample home bases are cryopreserved at -20oC for about 15 yearss. The experiment was carried out with different distance from liquid N to 70 % sucrose solution.Degree centigrades: UserskrishDesktopcontrol.jpgFigure: 3.

5.1: Negative ControlThe above figure contains 70 % sucrose sample that has been exposed to liquid N at different distances for approximately 4 hours, and cryopreserved it at -20oC for 5 to 15 yearss.Harmonizing to Morris check system above figure shows that after incubation for 5 yearss at -20EsC. The samples incorporating Petri dishes that has been exposed to merely liquid N at different distance ( i.e.

100cm, 200cm, and 300cm ) does non demo any alteration in the sample formation and same procedure was prolonged for 11 yearss and observed still no alterations has been observed, even after 15 yearss. This appears that taint is non occurred by the exposure of liquid N to the sucrose solution and it can be treated as negative control sample for experiment.

Consequences observed in Positive control:

Different phases of taint are showed in the undermentioned figures by taking 70 % sucrose solution sample in Petri dishes keeping at different distances to pollute liquid N with sucrose hemi-heptahydrate.Degree centigrades: UserskrishDesktop100cm.jpg C: UserskrishDesktop200cm.jpgFigure: 3.

5.2. a: 100 cm Figure: 3.

5.2. B: 200cmDegree centigrades: UserskrishDesktop300cm.jpgFigure: 3.5.2. degree Celsiuss: 300cmFigure: 3.

5.2. Observation of treated saccharose home bases after cryopreservationFigure: 3.5.2. a:Harmonizing to the above figure ‘a ‘ the sucrose sample solution in Petri dishes that has been exposed with liquid N incorporating sucrose hemi-heptahydrate at a distance of 100cm was incubated for 5-15days at temperature -20EsC shows more alterations in the samples with their taint by organizing white farinaceous constructions. As procedure prolonged for 5 more yearss that farinaceous constructions are increased and formed like a crystal construction are observed.Figure: 3.

5.2. B:Harmonizing to figure ‘b ‘ , the sucrose sample that exposed to contaminated liquid N at a distance of 200cm for 4 hours.

These exposed Petri dishes demoing little farinaceous constructions after cryopreservation at -20oC for 10 yearss. After 15 yearss these granules that are increased little more in figure and which are diamond crystals in construction. But these Numberss of crystals are bit less than other Petri dishes that are placed at 100cm distance to contaminated liquid N.Figure: 3.5.

2. degree Celsiuss:Harmonizing to figures ‘c ‘ the sample 70 % sucrose solution placed at a distance of 300cm to the contaminated liquid N with sucrose hemi heptahydrate. These Petri dishes are exposed to nitrogen bluess for 4 hours and incubated at -20oC for 15 yearss. After cryopreservation the crystal formation is seen in Petri dishes. Compare to other Petri dishes that are placed at 100cm and 200cm distance holding more figure of crystals than 300cm distance maintained with liquid N Dewar flask.If the sucrose sample temperature is get downs from -20oC to normal room temperature so these contaminated crystal construction are stable even in dissolved status. It can easy distinguish from the sample solution. These crystals are clearly observed under microscope.

Table: 1.

The mean per centum ( % ) of the taint at different distances:

Observation of taint per centum on check home bases after cryopreservation for 15 yearssExperiment100200cm300cmFirst experiment91 %76 %54 %Second experiment89 %72 %51 %Third experiment91 %67 %53 %Fourth experiment88 %75 %48 %Mean of Percentage89.75 %72.50 %51.

50Figure: 3.2. Percentage of taint at different distancesHarmonizing to the graph the samples that are exposed to liquid N incorporating sucrose hemi-heptahydrate at 100cm shows maximal crystal construction formation. That shows maximal taint. When distance additions to 200cm from N vapours the crystal formation in the sucrose sample is lessenings, if distance increased to 300cm from liquid N vapours the crystal formation is bit reduced in 70 % sucrose sample. That shows sucrose sample taint decreases as distance additions between liquid N Dewar flask and sucrose sample Petri dishes. But in control samples ( that is 70 % sucrose solution home bases exposed to the liquid N bluess at different distances ) are non demoing any crystal formation in it. After cryopreservation of control home bases for 15days besides there are no crystal formation in it.

So 0 % of crystals observed in control Petridishes. Harmonizing to the above consequence distance besides plays an of import function in cryopreservation for taint with liquid N.


This experiment has proved that the liquid N was chief beginning of taint during cryopreservation of 70 % sucrose solution at -20oC. The distance has besides played a major function in the taint during cryopreservation of sucrose solution. Other undertaking members has besides proved that the above hypothesis, purpose and aims. That the, Ravindhar has worked with different concentration of sucrose hemi heptahydrate, which contaminates the liquid N with the consequences, are demoing that the lower concentration of sucrose hemi heptahydrate contaminates less than the higher concentration of sucrose hemi heptahydrate in 70 % sucrose samples.

Another co-worker Srikanth was carried out the experiment with different exposure clip period to the contaminated liquid N, the consequences was obtained as the 70 % sucrose solution is more contaminated when the exposure clip was more, if the exposure clip is lessenings so the observation of taint besides decreased in 70 % sucrose solution. This experiment has showed that the potency of taint occurs by the liquid N bluess.Harmonizing to Morris ( 2005 ) an easy analysis was carried out the happening of taint during different phases of cryopreservation at -20oC.

In this check system it was observed the individual crystal in raised construction of sucrose hydrate. After 15 yearss cryopreservation, it was observed that a cardinal deep depression in sucrose hydrate crystals, increased in size of crystals and merger between the next sucrose hydrate crystals. In this check system the crystals are stable even after the sucrose solution dissolved ( Morris, 2009 ) .The liquid N vapor stage was acted as a go-between for the transportation of contaminations from the liquid N to the beginning. it was mentioned that the vapour stage of liquid N may be polluting, but the widespread was religions that the liquid N vapor stage was ‘safe ‘ for those preserved in unfastened or non – sealed flasks that are like cryovials are chiefly used for vitrification.

There was an issue rose that the exterior or which are non autoclaved cryocontainers will besides may necessarily doing taint during the dissolving methods or processs ( Fountain and Ralston. et.al. , 1997 ) .In the recognized recent yearss, the transportation of hepatitis B virus to the disease agony patients through the transplanted bone marrow cells was stocked in contaminated N ( Tedder and Zuckerman.

et.at. , 1995 ) . Similarly same type transportation of a viral agent to patient with the liquid N besides recognised in 1971 these viruses are concerns as the papovavirus ( Charles and Sire. 1971 ) .

There was a opportunity of likely simplex virus 1 and adenovirus type 2 was reassigning on cotton wool swabs into the Dewar flask and out of the Dewar flask was besides recognised ( Carroll et.al, 1995 ) .In the suspected embryos was preserved in liquid N incorporating Dewar flask or armored combat vehicles that has the opportunity to pollute with some of the murine pathogens, if these pathogens are entered by chance into the liquid N flasks or armored combat vehicles. The old surveies explained as the rating of cross – taint in between the cryotubes incorporating mouse 2 – cell Embryos and murine pathogens in liquid N ( Shigeru et.al. 2003 ) . If embryos are cryopreserved in liquid N and transmittal of the virus has been pollute the preserved cells or tissues.

Harmonizing to Bielanski the possibility of taint is happening in preserved experiments are comparatively more ( Bielanski, et.al, 2000 ) .Here research workers supposed that initial beginnings of taint of dry shippers are taking portion, i.e. shippers itself or the liquid N and they had a opportunity to inadvertently contaminate with microorganisms through the procedure of production methods, saving and transit.

This taint by and large involves in non – infective omnipresent micro – beings like which was identified in one of the dry shippers experiment ( Murray 2003 ) .

Chapter 4


The 70 % sucrose solution gets polluting when the liquid N bluess acts as beginning of taint and the sucrose hemi-heptahydrate Acts of the Apostless as polluting agent. While the distance between the liquid N flask and sucrose solution home bases is additions so the taint of sucrose samples are decreased. By detecting these consequences the liquid N besides plays an of import function in the formation of crystals during taint. This method of experiment proves the above hypothesis and purpose.

Future Work:

The exposure of liquid N to the 70 % sucrose solution with different clip intervals as besides proves the above hypothesis and purpose, and different concentrations used that sucrose hemi-heptahydrate to pollute the liquid N that is exposed to 70 % of sucrose solution check home bases besides proves the above hypothesis and purpose.